Theoretical calculations of electronic transitions in organic molecules for different modes of excitation allow one to predict the cross-section and fundamental anisotropy for multi-photon excitation. The fundamental anisotropy of NADH is near 0.54 for two-photon excitation from 690-740 nm, which is 46% higher than the value of 0.37 observed for one-photon excitation. This observation of a higher anisotropy with two-photon excitation was consistent with INDO/SDCI calculation of the one- and two-photon transition. NADH displays a low cross-section for two photon excitation, which suggest that fluorescence from NADH will be negligible for two-photon fluorescence microscopy and will not significantly interfere with observations of TPIF of other extrinsic probes used to label cells.